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Zinc finger protein 695 facilitates the proliferation of colorectal cancer cells through activation of the NEK2 and PI3K/Akt/mTOR signaling pathways

  • Authors:
    • Xiaowei Li
    • Xubin Li
    • Jing Wang
    • Mei Xue
    • Mengqiao Zhang
    • Junfang Shuai
    • Jie Zhang
    • Danyang Ding
    • Ye Wang
    • Shiyang Hou
    • Xiaoqian Chi
    • Haiying Sun
    • Qiang Gao
    • Chunbo Kang
  • View Affiliations / Copyright

    Affiliations: Department of Surgery, Center of Gastrointestinal Rehabilitation, Beijing Rehabilitation Hospital, Capital Medical University, Beijing 100144, P.R. China, Department of Gastroenterology and Hepatology, Center of Gastrointestinal Rehabilitation, Beijing Rehabilitation Hospital, Capital Medical University, Beijing 100144, P.R. China
    Copyright: © Li et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
  • Article Number: 116
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    Published online on: July 16, 2025
       https://doi.org/10.3892/or.2025.8949
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Abstract

Colorectal cancer (CRC) is the third most common malignant tumor and the second leading cause of cancer‑related deaths worldwide. Identifying driver genes in CRC development may provide clinical benefits for patients. Zinc finger protein 695 (ZNF695) is a nuclear protein with transcriptional regulatory activity, which has been implicated in tumor progression; however, the role of ZNF695 in CRC is unclear. The clinical relevance of ZNF695 and NIMA‑related kinase 2 (NEK2) in patients with CRC was analyzed based on The Cancer Genome Atlas database. Knockdown was performed by transfecting the cells with small interfering RNAs, whereas overexpression was induced by infecting the cells with a lentivirus. In addition, cell Counting Kit‑8, colony formation, cell cycle and apoptosis assays were carried out to assess the role of ZNF695 and NEK2 in CRC. Chromatin immunoprecipitation‑quantitative PCR (qPCR) and dual luciferase reporter assays were used to examine the transcriptional regulation of ZNF695 on the NEK2 gene. Reverse transcription‑qPCR and western blotting were applied to assess mRNA and protein abundance, respectively. The present study aimed to investigate the clinical relevance, contribution and downstream effects of ZNF695 in CRC. The results revealed that ZNF695 was upregulated in CRC tissues compared with that in non‑cancer tissues. CRC cells also expressed higher ZNF695 expression than normal cells. In vitro, knockdown of ZNF695 suppressed the proliferation of HCT‑8 cells; conversely, overexpression of ZNF695 promoted the malignancy of HT‑29 cells. Moreover, ZNF695 accelerated cell cycle progression and inhibited apoptosis in CRC cells. Mechanistically, it was revealed that ZNF695 upregulated the expression of NEK2 at both the mRNA and protein levels. Luciferase reporter assay demonstrated that ZNF695 enhanced the transcriptional activity of the NEK2 promoter. Furthermore, knockdown of NEK2 reversed the oncogenic function of ZNF695. Additionally, ZNF695 activated the PI3K/Akt/mTOR signaling pathway in CRC cells. Inhibition of this pathway with rapamycin resulted in higher cytotoxicity to CRC cells with ZNF695 overexpression, suggesting that elevated ZNF695 levels may increase the sensitivity of CRC cells to rapamycin. In summary, the current study identified ZNF695 as a tumor‑promoting protein in CRC through activation of the NEK2 and PI3K/Akt/mTOR signaling pathways. Targeting the NEK2 and PI3K/Akt/mTOR signaling pathways may therefore be a promising strategy for the treatment of patients with CRC and high ZNF695 expression.
View Figures

Figure 1

Clinical relevance of ZNF695 in
patients with CRC. (A) Transcript levels of ZNF695 in pan-cancer
was analyzed using UALCAN based on TCGA data. *P<0.05 vs. normal
group. (B) Transcript levels of ZNF695 in cancer (n=275) and
non-cancer (n=349) tissues of patients with CRC. *P<0.05. (C)
mRNA expression of ZNF695 in CRC tissues based on the individual
cancer stage and in non-cancer tissues was analyzed using Gene
Expression Profiling Interactive Analysis. ***P<0.001. (D)
Promoter methylation level of ZNF695 in cancer (n=313) and
non-cancer (n=37) tissues of patients with CRC was analyzed using
UALCAN. P>0.05. (E) Promoter methylation level of ZNF695 in in
CRC tissues based on the individual cancer stage and in non-cancer
tissues was analyzed using UALCAN. Normal vs. stage 2, P<0.05.
(F) Overall survival of patients with CRC who were divided into the
ZNF695 high expression group (n=406) and the low expression group
(n=655) was analyzed from Kaplan Meier plotter. P=0.065. (G) mRNA
expression levels of ZNF695 were detected by reverse
transcription-quantitative PCR in NCM460, HT-29, HCT-116 and HCT-8
cells. *P<0.05, **P<0.01 vs. NCM460. BLCA, bladder urothelial
carcinoma; BRCA, breast invasive carcinoma; CESC, cervical squamous
cell carcinoma and endocervical adenocarcinoma; CHOL,
cholangiocarcinoma; CRC, colorectal cancer; ESCA, esophageal
carcinoma; GBM, glioblastoma; HNSC, head and neck squamous cell
carcinoma; KICH, kidney chromophobe; KIRC, kidney renal clear cell
carcinoma; KIRP, kidney renal papillary cell carcinoma; LIHC, liver
hepatocellular carcinoma; LUAD, lung adenocarcinoma; LUSC, lung
squamous cell carcinoma; PAAD, pancreatic adenocarcinoma; PRAD,
prostate adenocarcinoma; PCPG, pheochromocytoma and paraganglioma;
READ, rectum adenocarcinoma; SARC, sarcoma; SKCM, skin cutaneous
melanoma; THCA, thyroid carcinoma; THYM, thymoma; STAD, stomach
adenocarcinoma; UCEC, uterine corpus endometrial carcinoma; TCGA,
The Cancer Genome Atlas; ZNF695, zinc finger protein 695.

Figure 2

Overexpression of ZNF695 supports the
proliferation of CRC cells. (A) mRNA and protein levels of ZNF695
in siCtrl, siZNF695#1 and siZNF695#2 HCT-8 cells were detected
using RT-qPCR and western blotting, respectively. **P<0.01 vs.
siCtrl group. (B) Proliferation of HCT-8 cells transfected with
siCtrl, siZNF695#1 and siZNF695#2 was determined using a CCK8
assay. *P<0.05, **P<0.01 vs. siCtrl group. (C) mRNA and
protein levels of ZNF695 in Ctrl and ZNF695-overexpressing HT-29
cells were detected using RT-qPCR and western blotting,
respectively. **P<0.01 vs. Ctrl group. (D) Proliferation of Ctrl
and ZNF695-overexpressing HT-29 cells was determined by CCK8 assay.
*P<0.05, **P<0.01 vs. Ctrl group. Colony formation was
assessed in (E) siCtrl, siZNF695#1 and siZNF695#2 HCT-8 cells, and
in Ctrl and ZNF695-overexpressing HT-29 cells. (F) Quantification
colony numbers *P<0.05, **P<0.01vs. siCtrl,
##P<0.01 vs. Ctrl group. CRC, colorectal cancer;
RT-qPCR, reverse transcription-quantitative PCR; CCK8, Cell
Counting Kit-8; ZNF695, zinc finger protein 695; si, small
interfering.

Figure 3

ZNF695 modulates the apoptosis and
cell cycle progression of CRC cells. (A) Cell cycle progression of
siCtrl, siZNF695#1 and siZNF695#2 HCT-8 cells was measured by
staining the cells with PI and via flow cytometric analysis. (B)
Cell cycle progression of Ctrl and ZNF695-overexpressing HT-29
cells was detected by staining the cells with PI and via flow
cytometric analysis. (C) Quantification of cell cycle results as
shown in A and B. *P<0.05 vs. siCtrl, #P<0.05 vs.
Ctrl group. (D) Apoptosis of siCtrl, siZNF695#1 and siZNF695#2
HCT-8 cells was assessed by staining the cells with Annexin V and
PI, followed by flow cytometric analysis. (E) Apoptosis of Ctrl and
ZNF695-overexpressing HT-29 cells was assessed by staining the
cells with Annexin V and PI, followed by flow cytometric analysis.
(F) Quantification of apoptosis results as shown in D and E.
*P<0.05 vs. siCtrl, #P<0.05 vs. Ctrl group. (G)
Caspase 3/7 activity was measured in HCT-8 cells transfected with
siCtrl, siZNF695#1 and siZNF695#2 and in HT-29 cells infected with
Ctrl and ZNF695-overexpressing lentiviruses. *P<0.05 vs. siCtrl,
#P<0.05 vs. Ctrl group. CRC, colorectal cancer;
ZNF695, zinc finger protein 695; si, small interfering.

Figure 4

ZNF695 affects the expression of p21
and Bcl-2. (A) Western blot analysis of p21, Bcl-2 and GAPDH in (A)
HCT-8 cells transfected with siCtrl, siZNF695#1 and siZNF695#2, and
in (B) HT-29 cells infected with Ctrl and ZNF695-overexpressing
lentiviruses. GAPDH served as internal control. ZNF695, zinc finger
protein 695; si, small interfering.

Figure 5

ZNF695 positively regulates the
expression of NEK2 in CRC cells. (A) List of genes that were
positively correlated with ZNF695 in patients with CRC based on The
Cancer Genome Atlas database. ***P<0.001. mRNA expression levels
of NEK2 were analyzed by reverse transcription-qPCR in (B) HCT-8
cells transfected with siCtrl, siZNF695#1 and siZNF695#2, and in
(C) HT-29 cells infected with Ctrl and ZNF695-overexpressing
lentiviruses. *P<0.05 vs. siCtrl group; **P<0.01 vs. Ctrl
group. Dual luciferase activity of the NEK2 gene promoter was
detected in (D) HCT-8 cells transfected with siCtrl, siZNF695#1 and
siZNF695#2, and in (E) HT-29 cells infected with Ctrl and
ZNF695-overexpressing lentiviruses. *P<0.05 vs. siCtrl group;
**P<0.01 vs. Ctrl group. (F) HT-29 cells were transfected with
ZNF695-Flag-pCDNA3.1 vectors, and the cells were subjected to
ChIP-qPCR experiments to assess whether ZNF695 interacted with the
promoter sequence of the NEK2 gene. Based on The Cancer Genome
Atlas database of patients with CRC. **P<0.01 vs. IgG group. (G)
there was a positive correlation between ZNF695 and NEK2 (R=0.62;
P<0.0001), and (H) a significant difference between the
transcript levels of NEK2 in cancer (n=275) and non-cancer (n=349)
tissues. *P<0.05. CRC, colorectal cancer; qPCR, quantitative
PCR; ChIP, chromatin immunoprecipitation; ZNF695, zinc finger
protein 695; si, small interfering; NEK2, NIMA-related kinase
2.

Figure 6

Overexpression of ZNF695 activates
the Akt/mTOR signaling pathway and dictates the sensitivity of CRC
cells to rapamycin. (A) Western blot analysis of NEK2, p-Akt, Akt,
p-S6, S6 and GAPDH in HCT-8 cells transfected with siCtrl,
siZNF695#1 and siZNF695#2. GAPDH served as an internal control. (B)
HCT-8 cells transfected with siCtrl, siZNF695#1 and siZNF695#2 were
treated with different concentrations of rapamycin for 48 h and
cell viability was determined by CCK8 assay. *P<0.05,
**P<0.01 vs. siCtrl group. (C) Growth inhibition rate in
response to different concentrations of rapamycin was calculated in
HCT-8 cells transfected with siCtrl, siZNF695#1 and siZNF695#2.
*P<0.05 vs. siCtrl group. (D) Western blot analysis of NEK2,
p-Akt, Akt, p-S6, S6 and GAPDH in HT-29 cells infected with Ctrl
and ZNF695-overexpressing lentiviruses. GAPDH served as an internal
control. (E) HT-29 cells infected with Ctrl and
ZNF695-overexpressing lentiviruses were treated with different
concentrations of rapamycin for 48 h and cell viability was
determined by CCK8 assay. *P<0.05, **P<0.01 vs. Ctrl group.
(F) Growth inhibition rate in response to different concentrations
of rapamycin was calculated in HT-29 cells infected with Ctrl and
ZNF695-overexpressing lentiviruses. *P<0.05 vs. Ctrl group. CRC,
colorectal cancer; CCK8, Cell Counting Kit-8; ZNF695, zinc finger
protein 695; si, small interfering; p-, phosphorylated; NEK2,
NIMA-related kinase 2; S6, ribosomal protein S6.

Figure 7

Knockdown of NEK2 rescues the
tumor-promoting function of ZNF695. (A) Protein levels of NEK2 in
Ctrl, ZNF695 and ZNF695 + siNEK2 HT-29 cells were detected by
western blotting. (B) Proliferation of Ctrl, ZNF695 and ZNF695 +
siNEK2 HT-29 cells was determined by Cell Counting Kit-8 assay.
*P<0.05, **P<0.01 vs. Ctrl, #P<0.05,
##P<0.01 vs. ZNF695. (C) Colony formation was assess
in Ctrl, ZNF695 and ZNF695 + siNEK2 HT-29 cells. *P<0.05 (Ctrl
vs. ZNF695), #P<0.05 (ZNF695 vs. ZNF695 + siNEK2).
(D) Caspase 3/7 activity was measured in Ctrl, ZNF695 and ZNF695 +
siNEK2 HT-29 cells. *P<0.05, (Ctrl vs. ZNF695),
#P<0.05 (ZNF695 vs. ZNF695 + siNEK2). CRC, colorectal
cancer; ZNF695, zinc finger protein 695; si, small interfering;
NEK2, NIMA-related kinase 2.

Figure 8

Diagram of the ZNF695 signaling
pathway in colorectal cancer tumorigenesis. ZNF695, zinc finger
protein 695; NEK2, NIMA-related kinase 2.
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Copy and paste a formatted citation
Spandidos Publications style
Li X, Li X, Wang J, Xue M, Zhang M, Shuai J, Zhang J, Ding D, Wang Y, Hou S, Hou S, et al: Zinc finger protein 695 facilitates the proliferation of colorectal cancer cells through activation of the NEK2 and PI3K/Akt/mTOR signaling pathways. Oncol Rep 54: 116, 2025.
APA
Li, X., Li, X., Wang, J., Xue, M., Zhang, M., Shuai, J. ... Kang, C. (2025). Zinc finger protein 695 facilitates the proliferation of colorectal cancer cells through activation of the NEK2 and PI3K/Akt/mTOR signaling pathways. Oncology Reports, 54, 116. https://doi.org/10.3892/or.2025.8949
MLA
Li, X., Li, X., Wang, J., Xue, M., Zhang, M., Shuai, J., Zhang, J., Ding, D., Wang, Y., Hou, S., Chi, X., Sun, H., Gao, Q., Kang, C."Zinc finger protein 695 facilitates the proliferation of colorectal cancer cells through activation of the NEK2 and PI3K/Akt/mTOR signaling pathways". Oncology Reports 54.4 (2025): 116.
Chicago
Li, X., Li, X., Wang, J., Xue, M., Zhang, M., Shuai, J., Zhang, J., Ding, D., Wang, Y., Hou, S., Chi, X., Sun, H., Gao, Q., Kang, C."Zinc finger protein 695 facilitates the proliferation of colorectal cancer cells through activation of the NEK2 and PI3K/Akt/mTOR signaling pathways". Oncology Reports 54, no. 4 (2025): 116. https://doi.org/10.3892/or.2025.8949
Copy and paste a formatted citation
x
Spandidos Publications style
Li X, Li X, Wang J, Xue M, Zhang M, Shuai J, Zhang J, Ding D, Wang Y, Hou S, Hou S, et al: Zinc finger protein 695 facilitates the proliferation of colorectal cancer cells through activation of the NEK2 and PI3K/Akt/mTOR signaling pathways. Oncol Rep 54: 116, 2025.
APA
Li, X., Li, X., Wang, J., Xue, M., Zhang, M., Shuai, J. ... Kang, C. (2025). Zinc finger protein 695 facilitates the proliferation of colorectal cancer cells through activation of the NEK2 and PI3K/Akt/mTOR signaling pathways. Oncology Reports, 54, 116. https://doi.org/10.3892/or.2025.8949
MLA
Li, X., Li, X., Wang, J., Xue, M., Zhang, M., Shuai, J., Zhang, J., Ding, D., Wang, Y., Hou, S., Chi, X., Sun, H., Gao, Q., Kang, C."Zinc finger protein 695 facilitates the proliferation of colorectal cancer cells through activation of the NEK2 and PI3K/Akt/mTOR signaling pathways". Oncology Reports 54.4 (2025): 116.
Chicago
Li, X., Li, X., Wang, J., Xue, M., Zhang, M., Shuai, J., Zhang, J., Ding, D., Wang, Y., Hou, S., Chi, X., Sun, H., Gao, Q., Kang, C."Zinc finger protein 695 facilitates the proliferation of colorectal cancer cells through activation of the NEK2 and PI3K/Akt/mTOR signaling pathways". Oncology Reports 54, no. 4 (2025): 116. https://doi.org/10.3892/or.2025.8949
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